The influence of structural variation, previously observed in a panel of V186.2 VH/V lambda 1‐expressing anti‐NP antibodies from the secondary response, on the affinity of these antibodies was examined by site‐specific mutagenesis and recombinant antibody construction. A tryptophan––leucine exchange at position 33 in the VH segment of all but one of the high‐affinity antibodies is the most frequently observed somatic mutation and by itself leads to a 10‐fold higher affinity; all other somatic exchanges are irrelevant for affinity selection. In the single case of a high‐affinity antibody without this common exchange, high affinity is mediated by a combination of mutations (including a one‐codon deletion) in VH and the particular D‐JH rearrangement carried by this antibody. The data indicate that the pattern of somatic diversification through hypermutation is shaped by affinity selection, but that only a single point mutation is available in the VH and the VL gene of lambda 1 chain‐bearing anti‐NP antibodies which by itself leads to an increase of hapten‐binding affinity. Based on the analysis of two secondary response antibodies from which somatic mutations in VH and VL have been eliminated, it is also concluded that the recruitment of B cell clones into the pathway of hypermutation involves a mechanism which is not based upon affinity differences towards the antigen.

Abstract. Rabbit antithrombin III (AT), purified by heparin-agarose, was labeled with iodine-131 by either the glucose oxidase-lactoperoxidase or iodine monochloride techniques. When intravenously injected, the disappearance of the '3'I-AT from plasma was characterized by rapid initial decreases, and three-exponential equations were required for best fit of the plasma disappearance curves. This rapid '3'I-AT removal was not caused by denaturation, as shown by comparison with results obtained when '3'I-AT was biologically screened (injected into a first rabbit, and then transferred 16 h later in whole plasma to a second for kinetic evaluation) before injection. Thus, the same rapid initial loss ofplasma '3'I-AT was observed with screened preparations, and the plasma fractional catabolic rates of 0.716±0.048 and 0.673±0.051 day-' for unscreened and screened '3'I-AT were not significantly different. These results support the hypothesis that a vascular-endothelial AT compartment is present in rabbit. The fractions of the total-body AT in the plasma, the vascular-endothelial and the extravascular compartments were 0.337±0.031, 0.178+0.056, and 0.485±0.069, respectively.Two three-compartment kinetic models are discussed. The first pictures AT as distributing independently between plasma and two other compartments, and the second sees AT as first passing to the vascular-endothelial compartment, and then directly into the extravascular

Carbon monoxide (CO) treatment improves pathogenic outcome of autoimmune diseases by promoting tolerance. However, the mechanism behind this protective tolerance is not yet defined. Here, we show in a transgenic mouse model for autoimmune diabetes that ex vivo gaseous CO (gCO)-treated DCs loaded with pancreatic β-cell peptides protect mice from disease. This protection is peptide-restricted, independent of IL-10 secretion by DCs and of CD4 + T cells. Although no differences were observed in autoreactive CD8 + T-cell function from gCO-treated versus untreated DC-immunized groups, gCO-treated DCs strongly inhibited accumulation of autoreactive CD8 + T cells in the pancreas. Interestingly, induction of β1-integrin was curtailed when CD8 + T cells were primed with gCO-treated DCs, and the capacity of these CD8 + T cells to lyse isolated islet was dramatically impaired. Thus, immunotherapy using CO-treated DCs appears to be an original strategy to control autoimmune disease.Keywords: Autoreactive CD8 + T cells r β1-integrin r Carbon monoxide r Dendritic cells r DiabetesAdditional supporting information may be found in the online version of this article at the publisher's web-site IntroductionAutoimmune diseases such as type 1 diabetes (T1D) are mainly chronic and impact the quality of life. Current therapy for T1D is based on daily injection of insulin; although insulin delivery Correspondence: Dr. Philippe Blancou e-mail: philippe.blancou@gmail.com methods are improving, this treatment adjusts glycemia imperfectly, resulting in hyperglycemic events ultimately leading to cardiovascular complications. Recent immunotherapeutic trials have focused on systemic immunoregulation protocols with success exemplified by anti-CD3 Ab treatment [1,2]. However, * These authors contributed equally to this work.C 2012 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim www.eji-journal.eu 210Thomas Simon et al. Eur. J. Immunol. 2013. 43: 209-218 by indiscriminately modifying the pathogenic autoimmune process together with beneficial immune responses, serious side effects may occur. An attractive alternative is to use Ag-specific approaches to selectively silence autoreactive T cells. As pivotal players in T-cell activation, DCs have been extensively studied to induce peptide-specific tolerance in numerous pathologies (reviewed in [3]). DCs also specifically regulate the T-cell expression of integrins, which are major molecules implicated in migration and function of effector T cells in nonlymphoid tissues (reviewed in [4]).A recent approach to induce tolerance has emerged using heme oxygenase-1 (HO-1) inducers or carbon monoxide (CO). Endogenous CO is only produced from the oxidative degradation of hemoproteins (e.g. hemoglobin, myoglobin, cytochrome) by the heme oxygenase enzymes, in particular HO-1 [5]. Up-regulation of the HO-1 pathway has a significant protective effect against induced [6] and spontaneous [7,8] autoimmune diseases, and allergy [9]. Several reports have also shown that overexpression of HO-1 and CO treatment can be ben...

As an aid for constructing recombinant antibodies we have developed pSV2gpt/neobased expression vectors which constitutively carry an enhancer S' of an immunoglobulin gene to be expressed. Advantage over conventional approaches is the greater choice of restriction sites available for recombining enhancerless V and C region genes allowing e.g. "forced cloning" schemes. In our laboratory these vectors have been used successfully to express various antibodies with manipulated V regions in stably transfected cell lines. Enhancer functions are supplied by the murine heavy chain enhancer (bases 2877-3559 of Genebank entry musigcdO7., Xba I-end of the original Xba 1/Eco RI-fragment converted to an Eco RI-end by A.Bothwell).The enhancer was cloned into pSV-VH ABam, a derivative of pSV-VtL4 (Ref.1, the internal BarnHi-fragments are deleted). The Eco RI-site at the vector-enhancer junction of pEnVH was deleted by partial Eco RI-digest, Klenow fill-up, Bam Hldigest, isolation of the fragments ex-RI tending In both directions from the Bam HI-site to the target Eco RI-site amp (symbolized by the arrows inside the orl // \\pEnVH diagram) and ligation. From o pSV-V.lam; L pEVH the indicated Pvu I/Eco RI-frag-6.7 kB VWJ ment can be transferred to other ex-BamHI pression vectors: this was done for gpt RI RI pSV2gpt and pSV2neo yielding pSVE2-RI clone Into gpt and pSVE2neo as well as for vec-RI tors carrying murine Ig constant re-Ig H Enhancer gion genes yielding pECy3, pECx and pECX (the letter "E" in the plasmid }|{{ pEnVH \ VDJ names denominates presence of the Ig 7EV 4k Do enhancer element described here). Re-BanHmI combinant V/C-regions are to be placed next to the unique Eco RI-site _ parHal RI-digest with a transcriptional orientation op-RI BaerHIdst posite to that of the dominant selec-Fragment Isolation + lIgaon table markers gpt or neo. RI Supported by Deutsche Forschungsgeg \Pvul RI meinschaft/SFB 74 through a grant to pEVH | V DJ L K.R.. 7.4 k5 E catte BamHI pSVEgpt, pSVEneo References: 1. Neuberger, M.N. (1983), EMBO J. 2, 1373-1378 354 Q 1. D r1 I Lllta, uTora, Lngan_ U IN L vresS LiMited, Uxtord, England.